Process for purification of chitosan by means of the salicylic acid salt thereof



United States PatentO PROCESS FOR PURIFICATIGN F CHITOSAN BY lgglANs OFTHE SALICYLIC ACID SALT THERE- John Doczi, Morristown, N. 3., assignorto Warner-Lam bert Pharmaceutical Company, Morris Plains, N. J., acorporation No Drawing. Application October 9, 1953, Serial No. 385,285

Claims. (Cl. 260-211) This invention relates to a process ofpurification and fractionation of certain aminopolysaccharidepreparations.

The aminopolysaccharides which I use in the practice of my invention maybe derived from the naturally occurring acetamidopolysaccharide, chitin,which is obtained from the shells of various Crustacea by an extractionprocedure such, for example, as that described by H. Brach, BiochemischeZeitschrift, volume 38, page 475 (1912).

The aminopolysaccharides which I employ in the practice of my inventionare derived from chitin by deacetylation. Such deacetylation is attendedby a certain degree of depolymerization, andwhen' drastic reactionconditions such as prolonged reaction time are employed partialdeamination occurs. The material which is obtained by the deacetylationof chitin is termed chitosan, a substantially acetyl-free partiallydepolymerized chitin which may be partially deaminated and whichconsists of a mixture of components which differ essentially from oneanother only in molecular weight. The term chito san', as used in thisapplication includes both undeaminated chitosan, which has a nitrogencontent of 8.7% and partially deaminated chitosan, Whose nitrogencontent is appreciably less than the value 8.7%, of undeaminatedchitosan. The exact molecular structure of partially deaminated chitosanis not known at present,

Since chitosan has considerable usefulness, as for example, as anintermediate in the preparation of certain anticoagulants as describedin my co=pending application, Serial No. 308,946, filed September 10,1952, it has become desirable to find some means of effecting apurification of chitosan as well as a fractional separation thereof intoa number of substantially homogeneous fractions covering a wide range inmolecular weight. Although many attempts have previously been made toachieve this objective none has resulted in any appreciable measure ofsuccess. I

I have now discovered a process wherebychitosan' may be purified fromextraneous material which includes inorganic salts, proteinaceousmaterials and gums. The purification procedure consists of adding anexcess of a soluble salicylate, preferably an alkali metal salicylatesuch as sodium salicylate, to an aqueous solution of a chitosan saltfollowed by chilling the resulting mixture in an ice-water bathwhereupon the chitosan precipitates as the salicylic acid salt thereof.By soluble salicylate, I means salicylic acid and those salicylic saltswhich are more soluble in Water than chitosan salicylate. The highsolubility of the sodium salicylate makes it particularly useful in thisinvention. The precipitate is separated by centrifugation, redissolvedin water andthe resulting solution is filtered and adjusted to a pH ofabout 9, by the addition thereto of a water-soluble base such as sodiumhydroxide, ammonium hydroxide or diethylamine, and the'resultingprecipitate is collected, washed with a watermiscible organic solventand dried, yielding the desired chitosan in purified form. This methodof chitosan purification is illustrated by Example I, described below,and

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. 2' the results obtained therein aref summarized in Table I. It will benoted from Table I that the product of the process described in ExampleI was obtained in high yield and represents a substantial increase innitrogen content corresponding to a substantial increase in purity.

A more extensive purification process, and one which results in a highdegree of resolution, providing a sharp fractionation of the chitosan,consists of adding an excess of an alkali metal salicylate such as.sodium salicylate to an aqueous solution of a chitosan salt, gentlywarming to effect complete solution followed by gradual cooling instages, in a thermostatically controlled cooling bath. In this mannerfractions of chitosan salicylate separate at the various temperaturelevels in accordance with the solubility characteristics of p theindividual fractions. Each fraction is separately collected, redissolvedin Water, and treated with a base to liberate the corresponding chitosanbase fraction in the manner indicated above. This method of chitosanfractionation is illustrated by Example II described below, and theresults obtained are indicated in Table II. A further illustration ofthe procedure of fractionation of chitosan, performed in a mannersimilar to that described in Example II, is shown in Table III.

Thisfraction was obtained by neutralization of mother liquor offractio11(f).

It will be noted that in accordance with the process of invention thechitosan used as starting material was resolved into a succession offractions WhOS'e reduced ViC6Siti cdvei a Wide range and follow 21hOrdeTlY d6- creas'ing sequence, reduced viscosity being a conventionalparameter which is proportional to molecular weight. It will thereforebe appreciated that the process of my invention makes possible theresolution of a heterogeneous mixture of chitosans of differentmolecular weights into a number of fractions of increased homogeneity.Repeated experiments have shown that in the course of operation of thenew process the treated chitosan undergoes no appreciable degradation.

3 All reduced viscosity measurements were prerformed on 1% solutions ofchitosan in normal aqueousacetic acid at 30 0.);

1 This fraction was obtained by neutralization of mother liquor oftraction (d). r

7 In the two procedures discussed above and illustrated by Examples Iand II respectively, the various experimental conditions employed may bevaried over a reasonably wide range without detracting from the objectof my invention, which is to provide chitosan of increased purity and/orincreased homogeneity. Thus the acid used to form the salt of thestarting chitosan may, for example, be hydrochloric,- acetic, formicacid and the like; similarly the base used in regenerating the purifiedchitosan base from the salicylic acid salt thereof may be any organic orinorganic base provided that the strength of said 'baseis greater thanthat of chitosan itself (pKb', about 7.8). In this connection, it willbe understood that the utility'of salicylic acid is enhanced by raisingthe temperature to increase its solubility. In the course of myinvestigation of the process of my invention I have preferred to employdiethylamine as the said base inasmuch as it is a convenient reagent, ofadequate basic strength and which leaves no residual ash. Further, amongthe process variables the ratio of equivalents of salicylate reagent to'equivalents of chitosan, may also; vary widely and appears to controlthe product yields in the vicinity of the value of about 2 of saidratio, as shown in Table IV. In the interest of high process yields itis preferred to use values of said ratio of not less than 3.

TABLE IV Effect of sodium salicylatcchitosan ratio on yield of chitosansalicylate precipitate l Sample'Number.. 1 1 2 3 Equivalents of Sodiumsalicylate added per Equivalent of Chitosan 2.28 2.84 3. 41 Percent ofTheoretical Yield of Precipitated Chltosan salicylate 32. 3 72.1 I 89. 6

The following examples are illustrative of my invention: -EXAMPLE I "aran washed first with hot methanol until free of salicylate ion (bytheferric chloride test), then with ether and was finally dried at 70 C.under vacuum. The desired purified chitosan (13.3 g.) contained 8.30%nitrogen. The results of this experiment are summarized in Table I.

EXAMPLE II A sample of chitosan (6.0 g., reduced viscosity, 0.758) whichupon analysis was found to contain 8.43% N (hence the 6.0 g. of chitosanrepresent 0.0362 equivalent) was dissolved in normal aqueous acetic acid(37 cc.) and the resulting solution was diluted with water (100 cc.) andthen treated with sodium salicylate (17.9 g., 0.112 equivalent) andheated to C. until all suspended solid dissolved. The resulting clearsolution was allowed to cool gradually to 35 C. whereupon a precipitatebegan to separate. The temperature of the mixture was maintained at 35C. for one hour with continuous stirring and thereafter the precipitatewas collected by centrifugation. The mother liquor was cooled furtherand a number of additional fractions were separated in a similar manner(see Table II). Each of the aforesaid fractions, consisting of chitosansalicylate, were separately dissolved in water and decomposed bytreatment with 2 N methanolic diethylamine until the pH of the mixturewas about 9. In each case the liberated fraction of chitosan base wasthoroughly washed with hot methanol, then with ether and was finallydried at C. under vacuum. The various desired fractions of purifiedchitosan were thus obtained having the characteristics set forth inTable II.

I claim:

1. The method of recovering purified chitosan from crude chitosan whichcomprises forming an aqueous solution of an acid salt of crude chitosan,adding at least two equivalents of a soluble salicylate per equivalentof chitosan to said solution of chitosan salt, separating the resultingprecipitate of chitosan salicylate, decomposing said chitosan salicylatewith a base and separating the resulting purified regenerated chitosan.

2. The method set forth in claim 1, wherein said acid salt of chitosancomprises the acetate.

3. The method set forth in claim 1 wherein said base comprisesdiethylamine.

4. The method set forth in claim 1 wherein said solublesalicylate'cornprises an alkali metal salicylate.

5. The method of recovering purified chitosan from crude chitosan whichcomprises forming an aqueous solution of an acid salt of crude chitosan,adding at least two equivalents of a soluble salicylate per equivalentof chitosan to said solution of chitosan salt, warming the resultingmixture until a clearsolution results, chilling the solution to about 5C., separating the resulting precipitate of chitosan salicylate,decomposing said chitosan salicylate with a base and separating theresulting purified regenerated chitosan.

6. The method of recovering purified chitosan from an acid salt of crudechitosan, which comprises adding at least two equivalents of a solublesalicylate per equivalent of chitosan to an aqueous solution of saidchitosan salt, separatiugthe resulting precipitate of chitosansalicylate, decomposing saidchitosan salicylate with a base andseparating-the resulting purified regenerated chitosan.

0.35 equivalent) in water (560 cc.) and the resulting j The method offractionating chitosan into a plurality of fractions of increasedhomogeneity which comprises adding at least two equivalents of a solublesalicylate per equivalent of chitosan to an aqueous solution of an acidsalt of chitosan, warming the resulting mixture until substantiallyclear solution results, gradually cooling the solution to a series ofprogressively lower temperature levels, separating the fraction ofchitosan salicylate which precipitates at each of said temperaturelevels, separately decomposing each of said. fractions of chitosansalicylate filtered and the filtrate was adjusted to pH 8-9 with}.

"metha'nolic diethylamine. ,The resulting precipitate was with a baseand separating the resulting series of regenerated chitosan fractions ofvarying molecular weights.

6 8. The method set forth in claim 7, wherein said acid References Citedin the file of this patent salt of chitosan comprises the acetate. TETENTS 9. The method set forth in claim 7, wherein said base UNFTED STA SPA 19 1936 comprises diethylamine. 2,040,879 Rlgby May 10. The methodset forth in claim 7 wherein said 5 OTHER REFERENCES soluble salicylatecomprises an alkali metal salicylate. Loewy, Chem 4 609 (910%

1. THE METHOD OF RECOVERING PURIFFIED CHITOSAN FROM CRUDE CHITOSAN WHICHCOMPRISES FORMING AN AQUEOUS SOLUTION OF AN ACID SALT OF CRUDE CHITOSAN,ADDING AT LEAST TWO EQUIVALENTS OF A SOLUBLE SALICYLATE PER EQUIVALENTOF CHITOSAN TO SAID SOLUTION OF CHITOSAN SALT, SEPARATING THE RESULTINGPRECIPITATE OF CHITOSAN SALICYLATE, DECOMPOSING SAID CHITOSAN SALICYLATEWITH A BASE AND SEPARATING THE RESULTING PURIFIED REGENERATED CHITOSAN.7. THE METHOD OF FRACTIONATING CHITOSAN INTO A PLURALITY OF FRACTIONS OFINCREASED HOMOGENEITY WHICH COMPRISES ADDING AT LEAST TWO EQUIVALENTS OFA SOLUBLE SALICYLATE PER EQUIVALENT OF CHITOSAN TO AN AQUEOUS SOLUTIONOF AN ACID SALT OF CHITOSAN, WARMING THE RESULTING MIXTURE UNTILSUBSTANTIALLY CLEAR SOLUTION RESULTS, GRADUALLY COOLING THE SOLUTION TOA SERIES OF PROGRESSIVELY LOWER TEMPERATURE LEVELS, SEPARATING THEFRACTION OF CHITOSAN SALICYLATE WHICH PRECIPITATES AT EACH OF SAIDTEMPERATURE LEVELS, SEPARATELY DECOMPOSING EACH OF SAID FRACTIONS OFCHITOSAN SALICYLATE WITH A BASE AND SEPARATING THE RESULTING SERIES OFREGENERATED CHITOSAN FRACTIONS OF VARYING MOLECULAR WEIGHTS.